Diazo derivatives of antibiotic x-537a

ABSTRACT

DERIVATIVES OF ANTIBIOTIC X-537A FORMED BY MODIFICATION OF THE CARBOXYL AND PHENOLIC GROUPS, SUBSTITUTION AT THE 5-POSITION OF THE AROMATIC RING AND REDUCTION OR OXIMATION OF THE KETONE ARE DESCRIBED. THESE DERIVATIVES RE USEFUL AS COCCODIOSTATIC AGENTS AND ANTIBACTERIAL AGENTS.

United States Patent 3,836,516 DIAZO DERIVATIVES 0F ANTIBIOTIC X-537AArthur Stempel, Teaneck, and John Westley, Mountain Lakes, NJ.,assignors to Hoffmann-La Roche Inc., Nutley, NJ.

No Drawing. Original application Aug. 18, 1969, Ser. No. 851,044, nowPatent No. 3,715,372. Divided and this application Oct. 24, 1972, Ser.No. 300,091

Int. Cl. A2311 1/17; A61k 21/00; C07c 113/04 US. Cl. 260-441 2 ClaimsABSTRACT OF THE DISCLOSURE Derivatives of antibiotic X537A formed bymodification of the carboxyl and phenolic groups, substitution at the-position of the aromatic ring and reduction or oximation of the ketoneare described. These derivatives are useful as coccidiostatic agents andantibacterial agents.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisionalapplication of co-pend ing application S.N. 851,044 filed Aug. 18, 1969and now US. Pat. 3,715,372 issued Feb. 6, 1973.

DESCRIPTION OF THE INVENTION This invention relates to novel derivativesof antibiotic X-537A and to methods of preparing these derivatives. Moreparticularly, the invention relates to derivatives of antibiotic X-537Aprepared by substitution on the phenyl ring of the antibiotic, orsubstitution at the ketone position of the antibiotic molecule. Thesenovel derivatives exhibit activity as coccidiostatic agents andantibacterial agents.

Antibiotic X-537A is the designation given to a crystalline antibioticproduced by a Streptomyces organism isolated from a sample of soilcollected at Hyde Park, Massachusetts. Lyophilized tubes of the culturebearing the laboratory designation X537 were deposited with the UnitedStates Department of Agriculture, Agricultural Research Service,Northern Utilization Research and Development Division, Peoria, 111. Theculture, given identification number NRRL 3382 by the AgriculturalResearch Service, has been made available to the public through NRRL.The culture is also available to the public from the InternationalCenter of Information in collaboration with WHO. in Belgium.

The antibiotic material, heretofore identified as antibiotic X-537A,upon laboratory analysis has been found to be3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6-oxo-7- [5 ethyl 3methyl-5-(ethyl-S-hydroxy-6-methyl-2- tetrahydropyranyl) 2tetrahydrofuryHheptyl]-salicylic acid, i.e., a compound of the formulaCOOH CH CH CzH5 3 O H CH Antibiotic X-5 37A is prepared by growing theStreptomyces organism in an aerated submerged culture, with the pH ofthe broth adjusted to about neutral, i.e., about 6.5 to 7.5. The mediumutilized contains a nitrogen source, such as yeast, a yeast derivedproduct, corn meal, bean meal and the like, with soybean meal being themost preferred; and a carbohydrate source, such as sugar, molasses andthe like, with brown sugar being the most preferred. The fermentationwas carried out at slightly elevated temperatures, i.e., between about25 and 35 C., with the preferred incubation temperature being about3,836,516 Patented Sept. 17, 1974 28 C. After an incubation of about 4to 6 days, the fermentation broth was filtered and the antibioticrecovered by extraction.

As indicated above, the present invention relates to novel derivativesof antibotic X-537A. The novel derivatives to which the inventionrelates are selected from the group consisting of compounds of theformula COORi CH3 CH3 CzHs CH3 C2115 R20 I O CH3 R3 OH cm wherein R isselected from the group consisting of hydrogen, lower alkyl, phenyl, andphenyl substituted by a member of the group consisting of halogen, nitroand lower alkyl; R is selected from the group consisting of hydrogen,lower alkyl, lower alkanoyl,

C00 CH CH3 CzHs CH:

CgH5 R20 III wherein R and 'R, are as described above and thepharmaceutically acceptable salts of the compounds of formula II.

As used herein, the term lower alkyl denotes straight or branched chainhydrocarbon groups containing from 1 to 7 carbon atoms inclusive, suchas methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and the like,with groups containing from 1 to 4 carbon atoms being preferred. Theterm lower alkanoyl includes the acyl residue of lower alkanoic acids,preferably containing from 2 t0 4 carbon atoms, for example, acetyl,propionyl, and the like. The term halogen includes all four formsthereof, that is fluorine, chlorine, bromine and iodine.

Representative of the compounds of formulae II and III are:

3-Methyl-6- [4,6-dihydroxy3,5-dimethyl-7-ethyl-7- [5- ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-2- tetrahydropyranyl)-2-tetrahydrofuryl] heptyl] salicylic acid;

( -3-Methyl-6- [7-ethyl-4-hydroxy-6-hydroxyimino- 3,5 -dimethyl-7- 5-ethyl-3 methyl-5 (5 -ethy1-5 -hydroxy-6-methyl-2-tetrahydro-pyranyl)-2-tetrahydrofuryl] heptylJsalicylic acid,sodium salt;

( -3 -Methyl-6- [7-ethyl-4-hydroxy-6-hydroxyimino-3 ,5-

dimethy1-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6methyl-Z-tetrahydro-pyr anyl) -2-tetrahydrofuryl] heptyl] salicylicacid, hydroxylammonium salt;

( -3 Methy1-6-{7-ethyl-4-hyd.roxy-6-hydroxyimino-3,5-dimethyl-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-Z-tetrahydro-pyranyl)-2-tetrahydrofuryl] heptyl}salicylic acid,sodium salt;

33-Methyl-6-{7-ethyl-4-hydroxy-6-hydroxyimiuo-3,S-dimethyl-7-[5-ethyl-3-methyl-5-(S-ethyl-S-hydroXy-dmethyl-2-tetrahydropyranyl) -2-tetrahydrofuryl] heptyl} salicylic acid,methyl ester, isomer A;3-Methyl-6-{7-ethyl-4-hydroxy-6-hydroxyimino-3,5-dimethyl-7-[5-ethyl-3-methyl-5-(S-ethyl-S-hydroxy-G-methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl] heptyl}salicylic acidmethyl ester, isomer B; 3Methyl-6-{7-ethyl-4-hydroxy-3,5-dimethy1-6-oxo-7-[5-ethyl-3-methyl-5-(S-ethyl-S-hydroxy-6-methyl-2-tetrahydro pyranyl) -2-tetrahydrof uryl] heptyl}acetyl salicylic acid, methyl ester;2-Methoxy-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl-6- xo-7- -ethy'l-3-methyl-5- S-ethyl-S-hydroxy- 6-methyl-Z-tetrahydropyranyl)-2-tetrahydrofury1] heptyl}benzoic acid, methyl ester;Ammonium-3-methy1-2-(Z-methyl-propionyloxy)-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroXy-6-methyl-2tetrahydropyranyl)l-tetrahydrofuryl]heptyl}benzoate;Ammonium-3-methyl2-nicotinoy1oxy-6-{7-ethyl-4-hydr0Xy-3,5-dimethyl-6-oxo-7-[S-ethyl-3 -methyl-5- 5- ethyl-S hydroxy-6-methyl-Z-tetrahydropyranyl)-2- tetrahydrofuryl]heptyl}benzoate;5-Nitro-3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethy1-6-oxo-7-[5-ethyl-3-methyl-5-(S-ethyl-5-hydroxy-6-methyl- Z-tetrahydropyranyl) -2-tetrahydrofuryl] heptyl} salicylic acid, sodiumsalt; 5-Amino-3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6- oxo-7-[5-ethyl-3-methyl-5- (S-ethyl-S-hydroxy-6-methyl- Z-tetrahydropyranyl)-2- tetr ahydrofuryl] heptyl} salicylic acid;5-Acetamido-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oxo-7-[5-ethyl-3-methyl-5- (5-ethyl-5-hydroxy-6- me thyI-Z-tetrahydropyranyl)-2-tetrahydr0furyl] hep tyl} salicylic acid;5-Acetamido-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oXo-7-[5-ethyl-3 -methyl- 5- (5-ethyl-S-hydroxy-6- methyl-Z-tetrahydropyranyl)-2-tetrahydrofu ryl] hep tyl} acetyl salicylic acid;5-Diazo-3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6- oxo-7-[5-ethyl-3-methyl-5- S-ethyl-5-hydroxy-6-methy1- Z-tetrahydropyranyl)-2-tetrahydrofuryl] heptyl} salicylic acid;5-Phenylazo-3-methyl-6-{7-ethyl-4-hydroXy-3,S-dimethyl- 6-oxo-7-5-ethy1-3-methyl-5- 5-ethyl-5-hydroxy-6- me thyl-2-tetr ahydropyranyl)-2-te tr ahydrof uryl] he p tyl} salicylic acid;2-Benzoyloxy-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl-6-oxo-7-[5-ethyl-3-methyl-5- (S-ethyl-S-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}benzoic acid.

A preferred group of compounds falling within the scope of formula IIare those having the formula COOR1 CH CH C2115 CH zHs CzH5 O O OH H 0CH3 II-a wherein R -R are as described above and the pharmaceuticallyacceptable salts thereof.

A most preferred group of compounds falling within wherein R is hydrogenor lower alkanoyl, R is hydrogen or halogen, and at least one of R or Ris other than hydrogen, and the pharmaceutically acceptable saltsthereof.

Representative of the compounds of formula II-b are:

3-Methyl-6-{7-ethyl 4 hydroxy 3,5 dimethyl-6-oxo-7- [S-ethyl 3methy1-5-( 5 ethyl-5-hydroxy-6-methyl-2- tetrahydropyranyl) 2tetrahydrofuryl]heptyl}acetyl salicylic acid, sodium salt;

3-Methyl 2 propionyloxy-6-{7-ethyl-4-hydroxy-3,S-dimethyl 6 oxo 7[S-ethyl-3-methyl-5-(S-ethyl-S-hydroxy 6methyl-Z-tetrahydropyranyl)-2tetrahydrofuryl]heptyl}benzoic acid, sodiumsalt;

Z-Butyryloxy 3 methyl 6 {7 ethyl-4-hydroxy-3,5-dimethyl 6 oxo 7[5-ethyl-3-methyl-5-(S-ethyl-S-hydroxy '6methyl-Z-tetrahydropyranyl)-2tetrahydro furyl]heptyl}benzoic acid,sodium salt;

S-Bromo 3 methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oxo-7-[5-etl1yl 3methyl 5 (5-ethyl-5-hydroxy-6- methyl 2tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid, sodium salt;

5-Iod0 3 methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl-6- oxoJ-[S-ethyl 2methyl 5 (S-ethyl-S-hydroxy-G- methyl 2tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid, sodium salt;

S-Chloro 3 methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oxo-7-[5-ethyl 3methyl 5 (5-ethyl-5-hydroxy-6- methyl 2tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid;

S-Bromo 3 methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oxo-7-[5-ethyl 3methyl 5 (5-ethyl-5-hydroxy-6- methyl 2tetrahydropyranyl)-2tetrahydrofuryl]heptyl}acetyl salicylic acid, sodiumsalt.

Further, the present invention relates to dehydrated derivatives ofantibiotic X537A. These dehydration products may be represented by theformula C O 0 R1 CH3 CH3 C2115 CH3 C2H5 l cm, R20

l I o 0 OH 1130 n, H O has wherein R R are as described above, and thepharmaceutically acceptable salts thereof.

Representative of compounds of formula IV are:

The derivatives of antibiotic X537A form a variety of pharmaceuticallyacceptable salts. These salts are prepared from the free acid form ofthe antibiotic or its derivatives by methods Well known in the art; forexample, by washing the free acid in solution with a suitable base orsalt. Examples of such pharmaceutically acceptable basic substancescapable of forming salts for the purpose of the present inventioninclude alkali metal bases, such as sodium hydroxide, potassiumhydroxide, lithium hydroxide and the like; alkaline earth metal bases,such as calcium hydroxide, barium hydroxide and the like; and ammoniumhydroxide. Alkali metal or alkaline earth metal salts suitable forforming pharmaceutically accept able salts can include anions such ascarbonates, bicarbonates and sulfates.

The compounds of formulas II, III and IV may be prepared following avariety of procedures. The choice of procedure will depend upon the siteor sites upon the antibiotic molecule where substitution is to takeplace. For example, the compound of formula I or compounds of formulasII and IV above wherein R is hydrogen can be converted into thecorresponding compounds of formulas II or IV above wherein R is alkyl,phenyl and substituted phenyl by conventional esterifying techniques.Representative of compounds which can effect the esterification arelower alkanols, such as ethanol, methanol, propanol, and the like; andaromatic alcohols, such as phenol, p-halophenol, p-nitrophenol,o-nitrophenol, trimethoxyphenol and the like.

The compound of formula I or compounds of formulas II and IV wherein Rand/ or R is hydrogen can be converted into the corresponding compoundswherein R and/ or R is lower alkyl. Preferably, the substitution isaccomplished by dissolving a compound of formula I or a compound offormulas II or IV above wherein R and/or R is hydrogen in an inertorganic solvent, such as N,N- dimethylformamide. To the resultingsolution is added an alkylating agent such as a lower alkyl halide,e.g., methyliodide, ethyliodide, propyliodide and the like, or a diloweralkyl sulfate, e.g., dimethyl sulfate, diethyl sulfate and the like. Thereaction mixture is allowed to stand at a temperature in the range ofabout to about 50, most preferably at room temperature. Whenadvantageous, the reaction is effected in the presence of a catalyst,which may be a noble metal oxide, such as silver oxide and the like. Byproceeding according to this method, from a compound of formula I orcompounds of formulas II or IV above wherein R and/ or R is hydrogen,there can be obtained compounds which contain a lower alkyl group in Rand/or R A compound of formula I or compounds of formulas II or IV abovewhich bear hydrogen at R can be provided with a lower alkanoyl groupwith any conventional and conveniently available lower alkanoyl groupproviding agents. Representative of such lower alkanoyl group providingagents are acid anhydrides, such as acetic anhydride, propionicanhydride and the like, and acid chlorides, such as acetyl chloride,propionyl chloride, butyryl chloride and the like.

Compounds of formulas II or IV above wherein R is halogen can beprepared from the compound of formula I or a compound of formulas II orIV above wherein R is hydrogen by any conveniently availablehalogenating technique. Among the many procedures suitable for thispurpose there may be included bromination utilizing bromine,chlorination utilizing chlorine, or iodination utilizing iodinemono-chloride and the like.

Compounds of formulas II or IV above wherein R is nitro can be preparedby treating the compound of formula I or a compound of formulas II or IVabove wherein R is hydrogen with nitric acid in the presence of glacialacetic acid. The resulting nitro compound can be hydrogenated in thepresence of any suitable reducing system, such as one which includesRaney nickel, to yield the corresponding compound wherein R is amino. Itis, of course, to be understood that when a compound of formula IV aboveis involved, careful selection of the hydrogenation system should beeffected so as to reduce the nitro group at R while leaving the doublebond present in the heptenyl radical unaffected.

If desirable, the compounds of formula II wherein R is amino, preparedas described in the preceding paragraph, may be converted to thecorresponding diazonium salt of formula III above 'by treatment withnitrous acid followed by cuprous chloride. The formation of thediazonium salt is effected by first preparing a solution of a compoundof formula 11 above wherein R is amino in a dilute mineral acid, such asaqueous sulfuric acid, aqueous hydrochloric acid and the like. Thissolution is then treated with nitrous acid. Preferably, the nitrous acidis provided by adding to said solution an aqueous solution of an alkalimetal nitrite, preferably sodium nitrite. The nitrous acid treatment ispreferably carried out at or below room tempera 6 ture so that thereaction does not proceed too energetically. Thus, temperatures between-5 to 25 C. are preferred.

Compounds of formula II and IV above wherein R is amino can be convertedto the corresponding compounds ing compounds wherein R is loweralkylamino by conventional alkylation procedures. For example, thesodium salt of a compound of formulas II or IV wherein R is amino can bereacted with a molar equivalent of a lower alkyl halide to obtain acompound of formulas II or IV wherein R is lower alkylamino. For thepurposes of this process, lower alkyl halides such as methyl iodine,ethyl bromide, sulfates such as dimethyl sulfate, diethyl sulfate andthe like may be used.

In an alternate process, compounds of formulas II and IV above wherein Ris lower alkylamino can be prepared by reducing the nitro group ofcompounds of formulas II and IV above wherein R is nitro in the presenceof a lower alkyl aldehyde and preferably in the presence of ahydrogenation catalyst such as Raney Nickel.

Compounds of formulas II and IV above wherein R is amino can also beconverted into the corresponding compounds wherein R is lower alkylamidoby treating the corresponding compounds wherein R is amino with a loweralkanoyl group providing agent. Representative of lower alkanoyl groupproviding agents are acid anhydrides, such as acetic anhydride,propionic anhydride, butyric anhydride and the like, and acid chloridessuch as acetyl chloride, propionyl chloride, butyryl chloride and thelike. Conveniently, this reaction is effected in the presence of aninert organic solvent, such as pyridine and the like.

Compounds of formulas II or IV wherein R is a phenylazo group or asubstituted phenylazo group can be prepared from the compound of formulaI or a compound of formulas II or IV above wherein R is hydrogen bytreating the same with a diazonium salt obtained from aniline orsubstituted derivatives thereof such as p-chloroaniline, p-nitroaniline,p-lower alkylaniline, p-lower alkoxyaniline and the like. Preferably,this reaction is effected by adding the aniline or the substitutedderivative thereof to an aqueous solution of sodium nitrite whereby toform the diazonium salt and then adding the resulting solutioncontaining the diazonium salt to an alcoholic solution of the compoundof formula I or a compound of formulas II or IV wherein R is hydrogen.Conveniently, the reaction is effected in the cold, e.g., fromtemperatures of 10 to 15, most preferably from 5 to 5.

Compounds of formula II above wherein R is a =N-OH group can be preparedfrom the compound of formula I or a corresponding compound of formula IIabove wherein R is a group by treating the ketone containing compoundwith hydroxylamine hydrochloride in a molar excess. The procedure ispreferably conducted in the presence of pyridine.

The ketone group present in the compound of formula I or a compound offormula II wherein R is =0 may be reduced to the corresponding compoundof formula II wherein R is OI-I by treating the starting material withan alkali metal borohydride, such as sodium borohydride, potassiumborohydride and the like. Preferably, the reduction is effected in thepresence of an inert organic solvent. Among the inert organic solventssuitable for this purpose may be included lower alkanols, such asmethanol, ethanol, propanol, butanol and the like, ethers, such asdiethyl ether, methylethyl ether, dioxane and the like, and similar typeorganic solvents.

Compounds of formula IV can be prepared from the compound of formula Ior a compound of formula II above wherein R is =0 by treating saidcompound with a dehydrating agent. Dehydrating agents suitable for usein the preparation of the compounds of formula IV above according to onebroad process aspect of the present invention may be represented byinorganic bases such as alkali metal hydroxides, e.g., sodium hydroxide,potassium hydroxide, and lithium hydroxide, and alkali earth metalhydroxides such as calcium hydroxide, barium hydroxide and the like. Aninorganic alkali metal base such as sodium hydroxide is preferred.

In one embodiment of the dehydrating process, the compound of formula Ior a compound of formula II above wherein R is is added to an inertorganic solvent, such as p-dioxane. To the resulting reaction mixture isadded an aqueous solution of the dehydrating agent, preferably aqueoussodium hydroxide and the mixture is permitted to stand. Althoughtemperature is not a critical aspect of this process embodiment, it ispreferable to perform the dehydration at a temperature of from about 20to about 50, most preferably at room temperature. The mixture can bemaintained under such conditions for a period of one to several hours,with a duration of about seven hours being preferred. Thereafter, thedesired product can be isolated and purified from the resulting mixture,which contains both isomers, employing conventional techniques such asrecrystallization or chromatography.

The compounds of formulas II and IV, either in their crystalline form orin the form of their pharmaceutically acceptable salts, and thecompounds of formula III are useful for both the prophylaxis and therapyof coccidiosis in poultry. Coccidiosis is a disease caused by amicroscopic protozoal parasite belonging to the genus Eimeria. Thecompounds of the present invention are especially useful because oftheir high activity against single and multiple infections, that isinfections caused by a single species or by a combination of species.Following standard procedures for ascertaining coccidiostatic activity,the compounds of formulas II, III and IV were administered to chickensat varying levels in their feed, with the result that these compoundswere shown to be active coccidiostats. For example, compounds such as:

3-Methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6-oxo7-[5- ethyl-3 -methyl- 5-ethyl-5 -hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}acetyl salicylic acid,sodium salt;

3-Methyl-2-pr0pionyloxy-6-{7-ethyl-4-hydroxy-3,5- dimethyl-6-oxo-7-5-ethyl-3-methyl-5- S-ethyl-S-hydroxy-6-methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}benzoicacid, sodium salt;

2-Butyryloxy-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oxo-7-5-ethyl-3 -methyl-5- 5-ethyl-5-hydroxy-6- methyl-2-tetrahydropyranyl-2-tetrahydrofuryl] heptyl} benzoic acid, sodium salt; and

5-Bromo-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl- 6-oxo-7-[5-ethyl-3-methyl-5- 5-ethyl-5-hydroxy-6- methyl-Z-tetrahydropyranyl-2-tetrahydrofuryl]heptyl} salicylic acid, sodium salt showcoccidiostatic activity in chickens at a feed level of 0.03 percent.

Further, the compounds of formulas II and IV, either in theircrystalline form or in the form of their pharmaceutically acceptablesalts, and the compounds of formula III are useful as antibacterialagents. Following standard procedures for determining antibacterialactivity, compounds of the present invention such as:

( -3-methyl-6-{7-ethyl-4-hydroxyimino-3 ,5 -dimethyl- 7- 5-ethyl-3-methyl-5- S-ethyl-5-hydroxy-6-methyl-2- tetrahydropyranyl-2-tetrahydrofuryl] heptyl}salicylic acid, hydroxylammonium salt;

5-bromo-3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-Z-tetrahydropyranyl) -2-tetrahydrofuryl] heptyl} salicylic acid, sodiumsalt;

5-nitro-3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6- oxo-7- 5-ethyl-3-methyl-5 5 -ethyl-5 -hydroxy-6- methyl-2-tetrahydropyranyl-2-tetrahydrofuryl] -heptyl} salicylic acid, sodium salt; and

8 5-iodo-3-methyl-6-{7-ethyl-4-hydr0xy-3,5-dimethyl-6- oxo-7- 5-ethyl-2-methyl-5 (5 -ethyl-5 -hydroxy-6-methyl- Z-tetrahy dropyranyl-2-tetrahydrofuryl] heptyl} salicylic acid, sodium salt show in vitroactivity against E. Bacillus.

The invention is further illustrated by the following examples. Unlessotherwise indicated, all temperatures given are in degrees centigrade.

EXAMPLE 1 Preparation of 3-methyl-6{7-ethyl-4-hydroxy-3,S-dimethyl 6oxo-7-[5-ethyl-3-methyl-5(5-ethyl-5-hydroxy-6- methyl 2tetrahydropyranyl) 2 tetrahydrofuryl] heptyl} salicylic acid(crystalline antibiotic X-537A) The Streptomyces organism was grown inaerated submerged culture in shaken flasks. The pH of the broth wasadjusted by the addition of KOH solution to 6.5-7.5, then the broth wassterilized. A tank fermentation was used wherein a 5-10% inoculurnconsisting of 3 day old submerged growth from aerated bottles was usedin the tank. The medium contained 2% soybean flour, 2% brown sugar, 0.5K HPO The fermentation was carried out at 28 C., under positive airpressure, with air-flows of 5-10 cu. ft. of air per minute per 40 togallon liquid charge. The broth was harvested after 4 to 6 daysfermentation, filtered, and the antibiotic was recovered by extraction.The extraction was carried out as follows:

204 Liters of broth were filtered and the wet filter cake was suspendedin liters of butyl acetate and mixture was stirred overnight, at roomtemperature. The mixture was then filtered and the water layer wasseparated and discarded. The butyl acetate solution, assaying 30 millionBacillus E units, was concentrated in vacuo to 3 liters, washed with 10%sodium carbonate solution, and dried with anhydrous sodium sulfate.

On further concentration to 300 ml. and dilution with 350 ml. ofpetroleum ether (b.p. 50-60 (1.), 41 g. of solid material, assaying 25million Bacillus E units, separated. This solid material was thenextracted in a Soxhlet apparatus with 4 liters petroleum ether (b.p.50-60 C.) for 40 hours. The extract was taken to dryness in vacuo, thecrystalline residue suspended in petroleum ether and filtered, yielding24.49 g. of a mixture of the salt and free acid form of3-methyl-6{7-ethyl-4-hydroxy-3,S-dimethyl- 6 oxo 7[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6- methyl 2tetrahydropyranyl-Z-tetrahydrofuryl]heptyl} salicylic acid. The motherliquor of the solid yielded an additional 5.73 grams of the antibiotic.

After recrystallization from ether-petroleum ether, this material, whichcontained sodium, was dissolved in ether and washed with dilute sulfuricacid to convert it to the free acid. Removal of the ether left an oilyresidue which crystallized from ethanol to yield pure 3 methyl 6 {7-ethyl 4-hydroxy-3,5-dimethyl-6-oxo-7-[5-ethyl-3-methyl- 5 (5ethyl-5-hydroxy-6-methyl-Z-tetrahydropyranyl)-2 tetrahydrofuryHheptyl}salicylic acid. Several recrystal lizations from ethanol did not changethe melting point which remained unsharp at 100l09 C.

EXAMPLE 2 Preparation of 3-methyl-6-{7-ethyl-3,5-dimethyl-6-oxo-7- [5ethyl 3 methyl-5-(5-ethyl-5-hydroxy-6-methyl-2- tetrahydropyranyl) 2tetrahydrofuryl] 4-heptenyl} salicylic acid, sodium salt, Isomer A To asolution of 10 g. of crystalline antibiotic X-537A in 100 ml. ofp-dioxane was added 200 ml. of 10% aqueous sodium hydroxide and themixture stirred at room temperature. After 7 hours the mixture, whichhad separated into two phases, was extracted twice with an equal volumeof ether. The ether extracts were dried (Na SO and conentrated to aviscous oil which on standing partially crystallized. The crystals wereremoved by the addition of ether and filtration. Recrystallization of asample from ether gave colorless needles of 3-methyl- 9 6{7-ethyl-3,5-dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-S-hydroxy-6-methyl-2-tetrahydropyranyl) 2 tetrahydrofuryl1-4-heptenyl}-salicylic acid, sodium salt, Isomer A; m.p. 220, [(11 19.96(1%, CH OH).

EXAMPLE 3 Preparation of 3-methyl-6-{7-ethyl-3,5-dimethyl-6-oxo-7- ethyl3 methyl-5-(S-ethyl-S-hydroxy-6-methyl-2- tetrahydropyranyl) 2tetrahydrofuryll-4- heptenyl} salicylic acid, sodium salt, Isomer B Theether filtrate obtained in Example 2 was evaporated to dryness,dissolved in hexane and chromatographed on a florisil column (200 g.)using gradient elution from 2 l. hexane/ether (1:1) to 2 l.ether/acetone (1:1). The first fraction eluted was discarded. The secondfraction was a mixture, which on concentration and treatment with ethylacetate gave an additional yield of 3-methyl-6- {7 ethyl 3,5dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5- ethyl 5-hydroxy-fi-methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl]-'4-heptenyl}salicylic acid, sodium salt, Isomer A. Chromatography of the filtrate onsilica gel using gradient elution from methylene chloride to acetonegave the second component of the mixture as an oil, which crystallizedon standing to yield 3-methyl-6-{7-ethyl-3,5- dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-( 5-ethyl-5-hydroxy- 6 methyl 2tetrahydropyranyl)-2-tetrahydrofuryl]-4- heptenyl} salicylic acid,sodium salt, Isomer '13; mp. 125", [04 +27.64 (1%, CH OH).

EXAMPLE 4 Preparation of 5-bromo-3-methyl-6-{7-ethyl-3,S-dimethyl- 6 oxo7 [5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6- methyl 2 tetrahydropyranyl)2 tetrahydrofnryl]-4- heptenyl} salicylic acid, sodium salt The 1.34 g.of crystalline antibiotic X-537A in carbon disulfide (25 ml.) was addeddropwise a solution of bromine (0.12 ml.) in carbon disulfide (10 ml.).The reaction mixture was left overnight under a stream of nitrogen toremove the CS The residue which contained the free acid form of thedesired end product, was dissolved in ethyl acetate. After washingsuccessively with aqueous sodium bisulfite and saturated sodiumcarbonate, the ethyl acetate solution was dried -(Na SO and evaporatedunder reduced pressure. Crystallization from methylene chloride/hexanegave colorless needles of 5- bromo-3-methyl-6-{7-ethyl-3,5-dimethyl-6-oxo-7 [5 -ethyl- 3 methyl 5(5-ethyl-5-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]-4-heptenyl}salicylic acid, sodium salt, m.p. 235, +9.63 (1%, DMSO).

EXAMPLE "5 Preparation of 5 bromo-3 methyl-6-{7-ethyl-4-hydroxy- 3,5dimethyl 6 oxo-7-[5ethyl-3-methyl-5- (5-ethyl-5- hydroxy6-methyl-2-tetrahydropyranyl)-2-tetrahydrofury1]heptyl} salicylic acid,sodium salt To a solution of 1.53 g. of the sodium salt of antibioticX-537A in ml. carbon disulfide was added dropwise a solution of 0.41 ml.bromine 10 ml. carbon disulfide at -5 over 10 minutes. The solvent wasremoved under a stream of nitrogen at room temperature (approximately 2hours). The residue was purified by dissolving it in ethyl acetate andthe solution washed successively with aqueous sodium bisulfite andsodium carbonate. After drying *(Na S04), the solution was evaporatedunder reduced pressure and the residue crystallized from ethyl acetateto give 5-bromo-3-rnethyl-6-{7-ethyl-4-hydroxy- 3,5 dimethyl 6 oxo 7[5ethyl-3-methyl-5-(5-ethyl-5- hydroxy 6 methylZ-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl salicylic acid, sodiunisalt as colorless cubes, m.p. 185, [a] -7.36 (1% in methanol).

10 EXAMPLE 6 Preparation of 3-metl1yl-6-{4,6-dihydroxy-3,5-dimethyl-7-ethyl 7 [5 ethyl 3 methyl-5-(5-ethyl-5-hydroxy-6- methylZ-tetrahydropyranyl) 2 tetrahydrofuryl] heptyl} salicylic acid To asolution of 10 g. of crystalline antibiotic X-537A in 500 ml. ofabsolute ethanol was added 1.36 g. of NaBH The reaction was followed byassaying with thin layer chromatography (silica gel=CHCl /acetone 8:2)and the reaction was continued overnight after adding an additional 1.0g. of NaBH The following morning the solvent was removed under reducedpressure and the heavy oil diluted with methylene chloride, washed withdilute HCl, dried (Na SO and concentrated to a solid foam. The slightlypink solid was chromatographed on a 500 g. silica gel column elutingwith 21. hexane-methylene chloride (1:1), 2 l. methylene chloride 2 l.methylene chloride ether (1:1). The 250 ml. fractions were assayed byTLC and fractions 13-17 pooled, concentrated to a yelloW solid andrechromatographed on a g. florisil column eluting with a gradient fromhexane to acetone. The fractions containing3-methyl-6-{4,6-dihydroxy-3,5- dimethyl 7 ethyl 7 [5 ethyl3-methyl-5-(5-ethyl-5- hydroxy 6 methyl 2 tetrahydropyranyl) 2tetrahydrofuryl]heptyl} salicylic acid were pooled and concentrated to asmall volume from which pure 3-methyl- 6 {4,6 dihydroxy 3,5dimethyl-7-ethyl-7-[S-ethyl 3- methyl 5 (5ethyl-S-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid crystals were recovered, mp. 151-153". From the motherliquor, additional material was crystallized, [a] +0.83 (C: .60%, CHOH).

EXAMPLE 7 Preparation of 2-methoXy-3-methyl-6-{7-ethyl-4-hydr0xy- 3,5dimethyl- 6-ox0-7-[5-ethyl-3-methyl-5- (5-ethyl-5- hydroxy6-methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl} benzoic acid,methyl ester A solution of 5 g. of crystalline antibiotic X-537A in 50ml. of N,N-dimethyl formamide was stirred overnight with 6 g. of silveroxide and 50 ml. of methyl iodide. After assay (TLC silicagel=benzene/acetone, 9:1) an additional 5 g. of silver oxide and 10 ml.of methyl iodide was added. After 48 hours the solids were removed byfiltration and the filtrate diluted with water and methylene chloride.The solvent was separated, removed under reduced pressure and theremaining N,N-dimethyl formamide removed by water Washing of an ethersolution. The ether layer was chromatographed on a 140 g. florisilcolumn eluting with a gradient between hexane (1 liter) to acetone '(1liter). The fractions containing the product were pooled andconcentrated to a clear oil which could be distilled at and 0.05 mm.yielded pure 2-methoxy- 3 methyl 6{7-ethyl-4-hydroxy-3,S-dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-2-tetrahydropyranyl) 2tetrahydrofurylJheptyl} benzoic acid, methyl ester, 10.79 (CH OH, C: 1%

EXAMPLE 8 Preparation of 3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl 6oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-Z-tetrahydropyranyl)-2 tetrahydrofuryl]heptyl} aeetyl salicylicacid, sodium salt To a solution of 6.12 g. (10 mm.) of crystallineantibiotic X-S37A in 10 ml. dry pyridine was added 2 ml. aceticanhydride. After 2 hours, 10 g. of ice was added to the reactionsolution and the resulting mixture was washed into a separatory funnelwith ethyl acetate and 1N HCl. The ethyl acetate solution was washedwith 1N HCl until all the pyridine had been removed. The ethyl acetatesolution was then washed with saturated sodium carbonate solution, dried(Na SO and evaporated to dryness under reduced pressure. The residue wasdissolved in methylene chloride and the solution evaporated on the steambath with additions of hexane until crystallization started. Cooling togave 3-methyl-6-{7-ethyl- 4 hydroxy 3,5dimethyl-6-oxo-7-[5-ethyl-3-methyl-5- ethyl 5hydroxy-6-methyl-2-tetrahydropyrany])-2- tetrahydrofuryl]heptyl}acetylsalicylic acid, sodium salt as white needles, mp. 186-187", [011 18.75(1%, CH OH).

EXAMPLE 9 Preparation of ()-3-methyl-6-(7-ethyl-4-hydroxy-6-hydroxyimino3,5 dimethyl 7 [5-ethyl-3-methyl-5-t 5- ethyl5-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryflheptyl)salicylic acid, hydroxyl-ammonium salt A mixture of g. of crystallineantibiotic X537A and 5 g. of hydroxylarnine hydrochloride in 50 ml.ethanol containing 5 ml. pyridine was heated under reflux for 60 hours.The solution was evaporated to dryness and 5 ml. water added. The solidproduct was triturated and filtered, then recrystallized from aqueousethanol to give 3-methyl-6-(7-ethyl-4-hydroxy-6-hydroxyimino-3,5-dimethyl 7 [5 ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6- methylZ-tetrahydro-pyranyl)-2-tetrahydrofuryl] heptyl) salicylic acid,hydroxylammonium salt; m.p. 168170, M1 -12.34 (1%, DMSO).

EXAMPLE 10 Preparation of(+)-3-methyl-6-{7-ethyl-4-hydroxy-6-hydroxyimino 3,5 dimethyl 7[5-ethyl-3-methyl-5-(5- ethyl 5hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl] heptyl}salicylic acid, sodium salt; and 3methyl-6-{7-ethyl-4-hydroxy-6-hydroxyimino- 3,5 dimethyl -7 [5ethyl-3-methyl-5-(5-ethyl-5-hydroxy 6' methyl 2tetrahydropyranyl)-2-tetrahydrofuryHheptyl} salicylic acid, sodium saltA mixture of 10 g. of crystalline antibiotic X-537A and 5 g. ofhydroxylamine hydrochloride in 50 m1. ethanol containing 5 m1. pyridinewas heated under reflux for 60 hours. The solution was evaporated todryness and treated with a mixture of 1N HCl and ethyl acetate. Theethyl acetate layer was washed with 1N HCl until all the pyridine andhydroxylarnine had been removed. It was then treated with a saturatedaqueous solution of sodium carbonate, separated and dried (Na SO Afterevaporation to dryness, the residue was fractionally crystallized fromethanol. The first fraction was shown by TLC on silica gel usingbenzene-methanol (9:1) as solvent to be the sodium salt of the oximepreviously isolated in Example 9 as its hydroxylammonium salt.Recrystallization from cH Cl /hexane gave white prisms of ()-3methy1-6-{7-ethyl-4-hydroxy-6-hydroxylimino- 3,5 dimethyl7-[5-cthyl-3-methyl-5-(S-ethyl-S-hydroxy- 6-methyl-2-tetrahydropyranyl)Z-tetrahydrofuryllheptyl} salicylic acid, sodium salt, m.p. 173174, [0:1-1l.99

1%, methanol). The second crop (1.63 g.) isolated by fractionalcrystallization of the crude sodium salt of the oxime was found to be anisomer of ()-3-methyl-6- {7 ethyl 4hydroXy-6-hydroxyimino-3,5-dimethyl-7- [5ethyl-3-methyl-S-ethyl-S-hydroxy-6-methyl2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl} salicylic acid, sodium salt.Recrystallization from CH Cl /hexane gave white needles of3-methyl-6-{7-ethyl-4-hydroxy-6- hydroxyimino 3,5dimethyl-7-[5-ethyl-3-methyl-5-(5- ethyl 5hydroxy-6-methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylicacid, sodium salt, mp. 220 0, [0th, +27.16 (1%, methanol).

EXAMPLE 11 [Preparation of 3-methyl-6-{7-ethyl-4-hydroxy-6-hydroxyimino3,5 dimethyl-7-[5-ethyl-3-methyl5-(S-ethyl- 5 hydroxy 6methyl-2-tetrahydropyranyl)-2-tetrahydrofury}salicylic acid methylester, Isomer A A solution of 3.55 g. of (-)-3-methyl-6-{7-ethyl-4-hydroxy 6 hydroxyimino 3,5-dimethyl 7 [5-ethyl-3- methyl 5 (5ethyl-5-hydroxy6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid,sodium salt in ml. methylene chloride was treated with 1N HCl. Thesolvent layer was separated, washed with water and concentrated underreduced pressure to a solid. A portion (1.3 g.) of the solid, equal to2.15 millimole, was dissolved in ether and treated with 4.25 millimoleof an ethereal solution of diazomethane. At the end of one hour thesolvent was removed at reduced pressure leaving an oil which uponstanding under a high vacuum solidified to yield3-methyl-6-{7-ethyl-4-hydroxy-6-hydroxyimino 3,5 dimethyl 7[5-ethyl-3-methyl-5-(5 ethyl 5hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylicacid, methyl ester, isomer A; [011 +5.77 (1.03%, CH OH).

EXAMPLE 12 Preparation of 3-methyl-6-{7-ethyl-4-hydroxy-6-hyd1'oxyimino3,5 dimethyl-7-[5-ethyl-3-methyl-5-(5-ethyl-5- hydroxy 6methyl-2-tetrahydropyranyl)-2-tetrahy drofuryl]heptyl}salicylic acidmethyl ester, Isomer B A solution of 1.77 g. of(+)-3-methy1-6-{7-ethyl-4- hydroxy 6 hydroxyimino3,5-dimethyl-7-[5-ethyl-3- methyl 5(5-ethyl-5-hydroxy-6-methyl-Z-tetrahydropyranyl)-2-tetrahydrofury1]heptyl}salicylicacid, sodium salt in 100 ml. of methylene chloride was washed with 1NHCl. The solvent layer was separated, washed with water, andconcentrated at reduced pressure to a solid. A portion (1.48 g.) of thesolid, equal to 2.45 millimoles was dissolved in ether and treated with5 millimoles of a solution of diazomethane in ether. After one hour thesolvent was removed in vacuo leaving an oil which upon standing underhigh vacuum solidified to yield 3-methyl-6-{7-ethyl- 4 hydroxy 6hydroxyirnino-3,5-dimethyl-7-[5-ethyl3 methyl 5(5-ethyl-S-hydroxy-6-methyl-Z-tetrahydropyranyl) 2tetrahydrofuryl]heptyl}salicylic acid methyl ester, Isomer B; +3263(1.01%, CH OH).

EXAMPLE 13 Preparation of 5-iodo-3-methyl-6-{7-ethly-4-hydroxy-3,5-dimethyl 6 oxo-7-[5-ethyl-2-methyl-5-(S-ethyl-S-hydroxy 6methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid,sodium salt A solution of 1.8 g. of crystalline antibiotic X-537A in 50ml. of glacial acetic acid was treated with 980 mg. of iodinemonochloride (freshly distilled 97-99 fraction). The ICl was mixed with5 ml. of acetic acid and added to the reaction slowly over a period of15 minutes. After an additional 15 minutes, water (100 ml.) was slowlyadded and the mixture extracted with ether. The solvent layer wasseparated and washed successively with aqueous solutions of sodiumbisulfite, sodium bicarbonate and sodium carbonate. The solvent wasdried (Na SOLQ. After concentration and crystallization, the product5-iodo-3-methyl- 6 {7 ethyl-4-hydroxy-3,5-dimethyl-6-oxo-7-[5-ethyl-2-methyl 5 (5-ethyl-5-hydroxy-6-methyl-2-tetrahydropyranyl)Z-tetrahydrofuryl]heptyl}salicylic acid, sodium salt was recovered byfiltration. Recrystallization from ethyl acetate gave5-iodo-3-rnethyl-6-{7-ethy1-4-hydroxy- 3,5 dimethyl 6oxo-7-[5-ethyl-2-methyl-5-(5-ethyl-5- hydroxy 6methyl-Z-tetrahydropyranyl)-2-tetIahydrofuryl]heptyl} salicylic acid,sodium salt, m.p. 222.5223 [M -48.7 (1%, CHCl Alternatively5-iodo-3-methyl 6 {7-ethyl-4-hydroxy- 3,5 dimethyl-6 oxo 7[5-ethyl-2-methyl-5-(5-ethyl-5- hydroxy 6 methyl 2tetrahydropyranyl)-2-tetrahy drofuryHheptyl} salicylic acid, sodium saltcould be prepared by treatment of crystalline antibiotic X537A (1millimole) with iodine (1 millimole) and morpholine (3 millimoles). Thereaction usually was complete in 5 days and the product, purified bychromatography, was identical to the above.

'13 EXAMPLE 14 Preparation of 3 methyl 6 {7 ethyl-4-hydroxy-3,5-dimethyl 6 oxo 7 [5 ethyl-3-methyl-5-(S-ethyl- 5 hydroxy6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl} acetyl salicylicacid, methyl ester 1.344 g. (2 mmole) of 3-methyl-6-{7-ethyl-4-hydroxy-3,5 dimethyl-6-oxo-7-[5-ethyl-3-methyl 5 (5-ethyl-5- hydroxy 6methyl-Z-tetrahydropyranyl)-2-tetrahydrofuryl] heptyl}acetyl salicylicacid, sodium salt was treated with ether (30 ml.) and 1N HCl (30 ml.).The mixture was shaken in a separatory funnel until all the startingmaterial had dissolved. The ether layer was then washed twice with waterand treated with 2 ml. solution of ethereal diazomethane mmole). Afterdrying (Na SO the solution was evaporated to give 3-methyl-6-{7-ethyl-4-hydroxy 3,5 dimethyl 6 oxo-7-[5-ethyl- 3-methy1 -5(5-ethyl-5-hydroxy 6 methyl 2 tetrahydropyranyl) 2tetrahydrofuryl]heptyl}acetyl salicylic acid, methyl ester as an oil,+O.99 (1% in methanol).

EXAMPLE 15 Preparation of 5 bromo-3-methyl-6-{7-ethyl-4-hydroxy-3,5-dimethyl 6 oXo-7-[5-ethyl-3-methyl-5-(5-ethyl-5- hydroxy-6-methyl-2tetrahydropyranyl) 2 tetrahydrofuryl]heptyl} acetyl salicylic acid,sodium salt To a solution of 2.11 g. of the sodium salt of S-bromo- 3methyl-6-{7-ethyl 4 hydroxy-3,5-dimethyl-6-oxo-7- [5 ethyl-3-methyl 5 (5ethyl-S-hydroxy-6-methyl-2- tetrahydropyranyl) 2 tetrahydrofuryllheptyl}salicylic acid in 20 ml. of dry pyridine was added 0.66 ml. of aceticanhydride. After 4 hours at room temperature, the reaction mixture wasdiluted with ice water containing 40 ml. of HCl and ethyl acetate. Theethyl acetate was Separated, washed with IE HCl until the pyridine wasremoved and then washed with a saturated aqueous solution of sodiumcarbonate and dried over Na SO The ethyl acetate was evaporated to 25ml. from which 5- bromo-2-methyl {7-ethyl 4 hydroxy 3,5-dimethyl-6- oxo7-[5-ethyl-3-methyl-5 (5-ethyl-5-hydroxy-6-methyl- 2-tetrahydropyranyl)2 tetrahydrofuryl[heptyl} acetyl salicylic acid, sodium salt wasrecovered by filtration. M.p. 213215 [ab -3.5 (1%, methanol). Anadditional yield of the desired product was recovered from the motherliquor after crystallization from methylene chloride/hexane.

EXAMPLE 16 Preparation of 5 nitro-3-methyl-6 {7-ethyl-4-hydroxy-3,5-dimethyl 6 oxo 7 [S-ethyl 3 methyl-S-(S- ethyl 5 hydroxy 6methyl-2-tetrahydropyranyl)- Z-tetrahydrofuryl] heptyl}salicylic acid,sodium salt To a solution of 2 g. of crystalline antibiotic X-537A in 75ml. glacial acetic acid was added 1 ml. concentrated nitric acid andafter stirring for /i hour, an equal volume (75 ml.) of water was added.The resulting yellow amorphous precipitate was extracted with ether. Theether layer was washed with sodium carbonate until all the acetic acidhad been removed. After drying (Na SO the ether was removed underreduced pressure to give 2.4 g. of a yellow solid. Treatment withmethylene chloride followed by filtration gave a crystalline productwhich was discarded. The methylene chloride filtrate was concentratedunder reduced pressure and chromatographed on a silica (20 g.) columnusing methylene chloride containing 1% methanol as eluant. The firstfraction eluted gave 5 nitro-3-rnethyl-6 {7-ethyl-4-hydroxy-3,5-dimethyl 6 oxo-7-[5 ethyl-3-methyl 5 (S-ethyl-S-hydroxy-6-methyl-2-tetrahydropyranyl) 2 tetrahydrofuryl]heptyl}salicylic acid, sodium salt which on recrystallization frommethylene chloride/hexane gave pale yellow plates, m.p. 214-215, [u]98.05" (1%, DMSO).

14 EXAMPLE 17 Preparation of 3 methyl-2-propionyloxy-6 (7-ethyl-4-hydroxy 3,5 dimethyl-6-oxo-7-(5-ethyl 3 methyl- S-(S-ethylS-hydroxy-6-methyl-2-tetrahydropyranyl)-Z-tetrahydrofuryl)heptyl)benzoic acid, sodium salt To a solution of 3.0g. (49 mmoles) of the crystalline antibiotic X-537A in 75 ml. ofpyridine, 0.7 ml. (54 mmoles) of propionic anhydride. was added and themixture kept at room temperature for 16 hours. Most of the solvent wasthen removed by concentration at reduced pressure below 35. The residuewas dissolved in methylene chloride, washed with dilute hydrochloricacid, dilute sodium carbonate and water and dried (Na SO Solvent wasthen removed by distillation under reduced pressure. The residuecrystallized from hexane to yield 3 methyl-2-propionyloxy-6-{7-ethyl 4hydroxy 3,5- dimethyl 6 oxo-7-[5-ethyl 3 methyl-S-(S ethyl-5- hydroxy 6methyl-2 tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}benzoic acid,sodium salt; m.p. 193195. Recrystallization did not alter the meltingpoint; -20.3 (C=1.3, MeOH).

EXAMPLE 18 In a similar fashion as that disclosed in Example 17, thefollowing compounds may be prepared from the indicated startingmaterials:

Z-butyryloxy 3 methyl-6-{7-ethyl 4 hydroxy 3,5- dimethyl-6-oxo-7 [5ethyl-3-methyl 5 (5 ethyl-5- hydroxy-6-methyl-2-tetrahydropyranyl) 2tetrahydrofuryl]heptyl}benzoic acid, sodium salt, m.p. 191193, [M -20.1(0:1, MeOH); from 3.0 g. of crystalline antibiotic X-537A and 0.88 ml.of butyric anhydride.

2-benzoyloxy 3 methyl-6-{7-ethyl 4 hydroxy-3,5- dimethyl 6 0x0 7[5-ethyl-3-methyl-5-(S-ethyl 5- hydroxy-6-methyI-Z-tetrahydropyranyl) 2tetrahydrofuryl]heptyl}benzoic acid, sodium salt, m.p. 177-180; fromcrystalline antibiotic X-537A and benzoic anhydride.

EXAMPLE 19 Preparation of the Ammonium Salt of Antibiotic X-537A 15.0 g.of the sodium salt of antibiotic X537A was dissolved in ethyl acetate(500 ml.) and washed three times with water, and three times with 200ml. portions of 2.8% ammonium hydroxide solution. Removal of solventfrom the ethyl acetate phase left a residue which was crystallized frommethylene chloride/hexane to afford white crystals of the ammonium saltof antibiotic X-537A, m.p. 195-196.5 (dec.), 9 s0.s4 (C=1.3 inmethanol). Concentration of the mother licuors afforded additionalmaterial, m.p. 194-196.

EXAMPLE 20 Preparation of Sodium Salt of Antibiotic X-537A The sodiumsalt was prepared by shaking an ether solution of the free acid ofantibiotic X-537A with aqueous sodium carbonate. The salt which remainedin the ether was recrystallized twice from benzene-ligroin and dried. Ithad a melting point, taken in an open capillary tube of 191-192 C. dec.

EXAMPLE 21 Preparation of the Lithium Salt of Antibiotic X-537A 5.0 g.of the sodium salt of antibiotic X-537A dissolved in ethyl acetate (200ml.). The solution was washed in turn with ml. portions of l Nhydrochloric acid (twice), water (twice), barium hydroxide solution(twice) and lithium sulfate solution (twice). A white solid wasrecovered by evaporation of the organic phase, and on recrystallizationfrom methylene chloride/hexane yielded the lithium salt as whitecrystals, m.p. 161162 (dec.), 8.27 (0:123 in methanol).

15 EXAMPLE 22 Preparation of 3-methyl-2-propionyloxy-6 {7 ethyl 4-Hydroxy-3,5-dimethyl-6-oxo-7-I5-ethyl-3 methyl 5-(S-ethyl-5-hydroxy-6-methyl 2 tetrahydropyranyl)-Z-tetrahydrofuryl]heptyl}benzoic acid, sodium salt 6.12 g. of the sodiumsalt of antibiotic X-537A was dissolved in pyridine 10 ml.) by warmingslightly. The solution was cooled in an ice bath and treated withpropionyl chloride, then allowed to stand at room temperature for 2hours. The mixture was poured into water, extracted with ethyl acetate,and the organic phase washed in succession with IE hydrochloric acid(several times) and sodium carbonate solution (twice). Evaporation ofthe ethyl acetate solution left a residue which was crystallized frommethylene chloride/hexane to afl'ord 3-methyl-2-propionyloxy-6-(7 ethyl4 hydroxy 3,5- dimethyl-6-oxo-7-(5-ethyl-3-methyl 5 (5 ethyl 5-hydroxy-6-methyl-2-tetrahydropyranyl) 2 tetrahydrofuryl)heptyl)benzoicacid, sodium salt, m.p. 185-1865 20.00 (C: 1.00 in methanol).

EXAMPLE 23 Preparation of ammonium 3-methyl-2-(2 methylpropionyloxy)-6-{7-ethyl-4-hydroxy-3,5-dimethyl-6 oxo-7-[5-ethyl-3-methyl-5-(S-ethyl-S-hydroxy 6 methyl- 2 tetrahydropyranyl)Z tetrahydrofuryl] heptyl} benzoate A solution of the sodium salt ofantibiotic X-537A (6.12 g.) in pyridine (10 ml.) was cooled in an icebath and treated with isobutyric 'anhydride 1.74 g. in '3 ml. ofpyridine). After stirring at room temperature for 2 days, the mixturewas poured into water, extracted with ethyl acetate, and the organicphase washed with 1E hydrochloric acid and 2.8% ammonium hydroxide. Theresidue obtained on evaporation of the ethyl acetate solution wasammonium-3-methyl-2-(2 methylpropionyloxy)-6-{7-ethyl-4-hydroxy-3,S-dimethyl 6 oxo 7 [5-ethyl-3-methyl-5-(5 ethyl-5 hydroxy 6 methyl 2- tetrahydropyranyl) 2tetrahydrofuryl]heptyl}benzoate in solid form, m.p. approx. 98 (dec.),[011L325 23.33 (C=1.25 in methanol).

EXAMPLE 24 Preparation of ammonium-3-methyl-2-nicotinoyloxy 6-{7-ethyl-4=hydroxy-3,5-dimethyl-6-oxo-7-[5 ethyl 3-methyl-5-(S-ethyl-S-hydroxy-6-methyl 2tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}benzoate A solution of thesodium salt of antibiotic X-537A (6.12 g.) in pyridine (10 ml.) wasstirred at room temperature under nitrogen with 3 g. of nicotinoylchloride hydrochloride for 13 days. Extraction and purification as inExample 22 gave a yellow residue which resisted crystallization.Attempted crystallization from hexane gave a guru which was dried on aclay plate to an amorphous solid, m.p. 8192 (dec.) {:1 :3 -14.64 (C=1.04in methanol).

EXAMPLE 25 Preparation of S-phenylazo 3 methyl 6 {7 ethyl 4-hydroxy-3,5-di-methyl-6-oxo-7-[S-ethyl 3 methyl 5--(S-ethyl-S-hydroxy-6-methyI-Z-tetrahydropyranyl) 2-tetrahydrofuryflheptyl} salicylic acid -To a solution of 0.18 ml. ofaniline in 8 ml. of E HCl, cooled to 05 C. in an ice bath was slowlyadded 150 mg: of NaNO in water chilled to the same temperature. To theabove solution was added a cold methanolic solution of 550 mg. ofcrystalline antibiotic X-537A. The reaction mixture was kept at 3 to 5for 15 minutes and then alloWGCl t9 lDWly reach room temperature. At the16 end of 2 hours, the reaction mixture was diluted with Water andextracted with ethyl acetate. The solvent extract was Washed with asaturated solution of N CO dried over 'NaSO and concentrated to a solid.The solid was chromatographed on a 30 g. silica gel (Grade 62) columneluting with a gradient between 300 ml. methylene chloride and 500 ml.ether. The product was eluted in the first peak and crystallized frommethylene chloride/hexane, mp. 212-215", [M ---27.30 (0.586%, CH OH).

EXAMPLE 26 Preparation of 5-amino-3-methyl-6-{7-ethyl 4 hydroxy-3,5-dimethyl-6-oxo-7-[5-ethy1-3-methyl 5 (5 ethyl- S-hydroxy-G-methyl 2tetr-ahydropyranyl) 2 tetrahydrofuryl] -heptyl}salicylic acid A solutionof 5.0 g. (7.6 millimole) of 5-nitro-3-methyl-6-{7-ethyl-4-hydroxy-3,S-dimethyl-G-oxo 7 [5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl 2tetrahydropyranyl)-2-tetrahydrofuryl]-heptyl}salicylic acid in ethanol(450 ml.) was hydrogenated over 2 g. of Raneynickel with an uptake of22.2 mm. of hydrogen. The reaction was filtered (over N and 17.2 ml. ofE HCl was added. The solvent was removed under reduced pressure, and theresidue was crystallized from aqueous acetone to give5-amino-3-methyl-6-{7 ethyl 4 hydroxy-3,5-dimethyl-6-oxo-7-[5-ethyl-3-methyl 5 (5 ethyl 5- hydroxy 6 methyl 2tetrahydropynanyl) 2 tetrahydrofuryl]-heptyl}salicylic acid, m.p.223-225" [111 +17.92 (1%, MeOH).

EXAMPLE 27 Preparation of 3-methy1-6-{7-ethyl-4-hydroxy-3,5-dimethyl 6oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6 methyl 2tetrahydropyranyl) 2 tetrahydrofuryH- heptyl}salicylic acid, methylester To a solution of 6.2 g. of crystalline antibiotic X-537A in ml. ofmethylene chloride was added 4.6 g. of silver oxide and 14 g. of methyliodide. After stirring for 18 hours, the reaction mixture was filteredand the filtrate concentrated to 7.07 g. of a light yellow oil, whichwas decolorized with Darco, giving 6.09 g. of a colorless foam. Aportion (4 g.) of the foam was chromatographed on a florisil column g.)eluting with a gradient between methylene chloride (1 l.) to ether (1l.) and then ether (500 ml.) to acetone (500 ml.) Rechromatography ofthe slightly yellow oil, recovered from the florisil column, on a 75 g.silica gel column and eluting with a gradient between methylene chloride(1 l.) and 15% ether/methylene chloride gave 200 mg. of a clear viscousoil, Bp. d./0O5 mm. --7.19, (1.1%, CH OH).

EXAMPLE 28 Preparation of 5 acetamido 3-methyl-6-{7-ethyl-4-hydroxy 3,5dimethyl-6-oxo-7-[S-ethyl-3-methyl-5-(5- ethyl5-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}salicylic acid To a suspension of 1.21 g. (2mmole) of 5-amino3- methyl 6 {7-ethyl-4-hydroxy-3,5-dimethyl-6-oxo-7-[5-ethyl3-methyl-5-(S-ethyl-S-hydroxy-6-methyl-2-tetrahyhydroxyranyl)-2-tetrahydrofuryl]-heptyl}salicylicacid in 50 ml. glacial acetic acid was added 1 ml. acetic anhydride (-10mmoles). On warming the mixture to 70 on a steam bath, the aminedissolved and after a further half an hour at room temperature, thesolution was poured on to crushed ice. The mixture was carefully madealkaline with sodium carbonate solution and extracted twice with 400 m1.ether. After drying (NaS0 the ethereal solution was evaporated toapproximately 100 ml. The resulting microcrystalline product had mp.189-190", [aJ 17.45 (1%, MeOH).

17 EXAMPLE 29 A solution of 5-amino-3-methyl-6-{7-ethyl-4-hydroxy- 3,5dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl] heptyl}salicylic acid(1.28 g.) in 10 ml. methanol and 2 ml. of aqueous N HCl was cooled toand a chilled solution of 150 mg. of NaNO in 1 ml. water was slowlyadded. To the solution was added 320 mg. of CuCl in 1 ml. ofconcentrated HCl. The reaction mixture was allowed to slowly warm up toroom temperature and then diluted with water and extracted with ether.The ether extract was concentrated to a small volume and thecrystallized product was collected by filtration, m.p. 137-138. [or],-l3.5 (0.7%, CH O-H).

EXAMPLE 30 Preparation of acetamido 3-methyl-6-{7-ethyl-4-hydroxy 3,5dimethyl-6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]heptyl}acetylsalicylic acid To a solution of 5 amino-3-methyl-6-{7-ethyl-4-hydroxy3,5 dimethyl 6-oxo-7-[5-ethyl-3-methyl-5-(5- ethyl 5hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl]-heptyl}salicylicacid (1.21 g.) in 20 ml. of pyridine was added 0.56 ml. of aceticanhydride. At the end of 4 hours, the reaction was poured into 100 ml.ice water containing 20 ml. of concentrated HCl. The solution wasextracted with ether, twice, and the solvents concentrated to dryness.The residue was dissolved in ethyl acetate from which a semicrystallinematerial was recovered after addition of hexane. Recrystallization ofthis material from methylene chloride/ether gave the crystallineproduct, m.p. 190-192", l8.38 (C, 1.1%, CH OH).

EXAMPLE 31 Preparation of 5 chloro-3-methyl-6-{7-ethyl-4-hydroxy- 3,5dimethyl 6-oxo-7-[5-ethyl-3-methyl-5-(5-ethyl-5- hydroxy 6 methylZ-tetrahydropyranyl) -2-tetrahydrofuryl]heptyl}heptyl}salicylic acid Toa cold solution (-3") of 6.12 g. of the sodium salt of antibiotic X-537Ain 50 ml. of chloroform/3 ml. of carbon tetrachloride was slowly added34 ml. of carbon tetrachloride saturated with 10 millimoles of chlorinegas. The reaction was continued for /2 hour after which the solution wasfiltered and the filtrate treated with a saturated aqueous solution ofNa CO The solvent layer was dried over Na SO and concentrated to a smallvolume from which after addition of hexane the crystalline product wasrecovered. The crystalline material which had a tendency to incorporatethe solvent of crystallization was twice recrystallized from methylenechloride/ether and acetone/hexane, m.p. 183-185 [0;]; 44.31 (C, 0.99%,CHCl EXAMPLE 32 This example illustrates the utilization of arepresentative member of the compounds of the present invention as acoccidiostatic agent in an animal feed. In a manner similar to thatdescribed below, the remaining compounds encompassed by the presentinvention may also be incorporated as active coccidiostatic agents intoanimal feeds.

A medicated poultry feed intended as a starter feed for broilers isprepared by blending 0.005 percent by weight of 3 methyl6-{7-ethyl-4-hydroxy-3,5-dimethyl-6-oxo-7-[5- ethyl3-methyl-5-(5-ethyl-S-hydroxy-G-methyl-2-tetrahydropyranyl)2-tetrahydrofuryl]heptyl} acetyl salicylic acid, sodium salt in a basicpoultry ration consitsing of:

Corn meal, No. 2. yellow, ground 1123 Stabilized grease or vegetable oil60 Soybean oil meal (low fiber content 50% protein) 480 Corn Gluten meal50 Fish meal, antioxidant treated, 60% protein 30 Fish solubles, driedbasis 10 Meat and bone scraps, 50% protein Corn distillers driedsolubles 50 Alfalfa meal, 17% protein 100,000 A/lb. 30

Salt, iodized 5 Manganese sulfate, feed grade 0.75 Zinc carbonate oroxide 0.25 Ribofiavan, Grams 3 Vitamin B mg. 6 Calcium pantothenate,gms. 5 Niacin, gm 30 Stabilized vitamin A USP units 6,000,000 Vitamin DIC units 650,000 Vitamin E acetate, IU 5,000

Vitamin E (menadione sodium bisulfite) gms. 2

DL-methionine or hydroxy analog, pound 1 Antioxidant (ethoxyquin orbutylated hydroxy toluene) lb. 0.25

Similar feeds can be prepared containing the active ingredient 3methyl-6-(7-ethyl-4-hydroxy-3,5-dimethyl-6- oxo7-(5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl- 2 tetrahydropyranyl)Z-tetrahydrofuryl)heptyl) acetyl salicylic acid, sodium salt at otherconcentrations, for example containing from 0.001 percent to 0.0125percent by weight of said compound.

What is claimed is:

1. A compound of the formula 000 cm cm C2HJSCHS H3O B OH R4 wherein R isselected from the group consisting of hydrogen, lower alkyl, loweralkanoyl,

References Cited UNITED STATES PATENTS 3,501,568 3/1970 Haney et al.260-345.7 X 2,600,690 6/ 1952 Robbins 260141 X 2,852,503 9/1958 Long etal 260152 3,148,983 9/1964 Endermann et al. 260--l41 X FLOYD DALE HIGEL,Primary Examiner US. Cl. X.R.

